Method of cell cultures and device for implementing it

ABSTRACT

The invention generally provides a cell culture vessel having at least one first zone and at least one second zone, wherein the first zone is a transfer zone for a culture medium which essentially contains no cells and the second zone is a cell culture zone. The invention further includes methods utilizing the cell culture vessel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/EP2006/066981, filed Oct. 3, 2006, which in turn, claims thebenefit of Belgian patent application No. 2005/0483, filed Oct. 4, 2005.The disclosures of each of the aforementioned applications areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to culture of cells by means of a devicecomprising:

a culture vessel provided with a cover, in which there is situated atleast one first zone and at least one second zone, said first zone beinga transfer zone for the culture medium essentially free from cells andsaid second zone being a cell culture zone,

means of circulating the culture medium, allowing circulation of theculture medium through the culture zone, said culture zone comprising abottom wall and a top wall, each wall being provided with orificesallowing a transfer of culture medium essentially free from cells.

BACKGROUND OF THE INVENTION

Culture devices are known for example from the U.S. Pat. No. 5,501,971which describes a culture vessel in which there is situated a culturezone external to an internal medium transfer conduit. The cells aresituated in the external culture zone, which is a sort of basketcomprising carriers, and the culture medium flows from top to bottomthrough this culture zone. Next the medium is recovered in the bottompart of the culture zone, it is taken up by means of a mediumcirculation device through the above mentioned conduit into a top partof the culture vessel and then passes again through the culture zone.The U.S. Pat. No. 5,507,971 describes several alternatives, but themedium always passes through the culture zone from top to bottom.

One drawback of such a device is that it is not adapted to all types ofcell culture. Indeed this device is solely designed for cell culture onmicrocarriers in a fixed or close-packed bed, and is absolutely notsuitable for cell culture in suspension or on microcarriers in afluidised bed. Indeed, in the device of the U.S. Pat. No. 5,501,971, thecells in suspension without carriers or microcarriers or onnon-immobilised carriers have a tendency to sediment and to accumulatein the bottom of the culture zone since they are subject to gravity and,as the flow of medium through this culture zone takes place from top tobottom, this accumulation of cells in the bottom of the culture zone isall the greater. Consequently the cells are packed on top of one anotherand are in contact with each other and the nutriments in this zone arenot very accessible for the cells.

In the reactor of the U.S. Pat. No. 5,501,971, in the case of cultures,on non-immobilised carriers or microcarriers, of cells which are fragilespecies withstanding few stresses, the cells are subjected to the effectof gravity, to the descending flow of the medium and to the weight ofthe carriers, which enormously impairs the survival of the culture. Inaddition, an application for the culture in suspension is inconceivablein the bioreactor of the U.S. Pat. No. 5,501,971 since the cells insuspension can under no circumstances be subjected to cell to cellcontact. Indeed, if such contacts are present, the cells create proteinbonds between them and aggregate. Such aggregation creates cell deaththrough lack of oxygen and nutriment.

U.S. Pat. No. 5,270,207, discloses a device wherein culture medium flowsfrom the bottom to the top through the culture zone. The space inbetweenthe culture zone and the outer side wall of the device is filled withmedium flowing upwards. The medium undergoes gas exchange in the upperspace of the device whereafter it flows downwards again via acylindrical zone in the center of the device towards the bottom of thedevice. In such devices, only a limited amount of gas exchange can takeplace, which makes these less suitable for cell culturing method whereinextensive cell growth or metabolism takes place.

SUMMARY OF THE INVENTION

The aim of the invention is to mitigate the drawbacks of the prior artby procuring a culture device making it possible to cultivate both cellsin suspension and anchorage-dependent cells on carriers or microcarrierswhilst ensuring minimum stress on the cells and preventing thisaccumulation of cells in the bottom of the culture zone.

To solve this problem, a device as indicated at the beginning of thistext, is provided according to the invention, characterised in itsbroadest sense in that it also comprises at least one third and at leastone fourth zone, both being culture medium transfer zones essentiallyfree from cells, said third zone being in medium communication with thefirst and second zone and said fourth zone being in medium communicationwith the second zone (the culture zone) and with the first zone (themedium transfer zone) via the culture medium circulation means, and inthat the culture medium circulation means allow a circulation of theculture medium from bottom to top in said second culture zone.

More particularly said third zone (4), has an overflow (37) so that theculture medium overflows from the first culture medium transfer zone (3)to the third culture medium transfer zone (4).

According to a more particular embodiment the third zone is a zoneinternal to said second zone and external to said first zone and saidfourth zone is a zone external to said second zone

According to an alternative embodiment the third zone is a zone locatedentirely below the second zone and entirely above the first zone.

According to a particular embodiment a device according to the inventioncomprises flow redistributing elements in the third zone or closedregions in the bottom wall of the second zone for providing a homogenousflow of medium into said second zone.

Said fourth zone more particularly encloses a volume of gas consistingof the ambient atmosphere of the bioreactor and can also constitute anoxygenation zone for this culture medium.

Consequently, the medium which passes through the first medium transferzone from bottom to top reaches the top of the first medium transferzone and overflows into the third zone, which is in medium communicationwith the first zone, the medium flows downwards, subjected to the flowimposed by the circulation means, to gravity and to the communicatingvessels effect, passes through medium-passage orifices in the bottomwall of the second culture zone, next travels up again towards the topof the culture zone by a communicating vessels effect or by an effectdue to the medium circulation means, or by both, and then overflows intothe fourth medium transfer zone, which is in communication with thefirst medium transfer zone via the circulation means. The mediumcirculation means then once again take the medium to the top of thefirst medium transfer zone and the cycle recommences. Consequently, thecells which are situated in the culture zone travelled through frombottom to top by the culture medium benefit from a gravity effect partlycounteracted by the flow of medium. The flow allows better dispersion ofcells alone or on microcarriers in the culture zone and the harmfulstresses for them are reduced.

When the terms cells on carriers or on microcarriers are used, it mustbe understood that the carriers can be in a fixed or packed bed or in afluidised bed.

Likewise, when the terms “cell culture” or “cells” are used, it must beunderstood in particular that animal cells are being referred to,whether it be for viral production, proteins or other recombinantproducts, cellular metabolites, a culture of tissue cells (possibly onthree dimensional carriers), stem cells or bacteria or yeasts.

It will be easily understood by the skilled in the art that cells oncarriers or microcarriers suffer less from packing, in particular in thecase of a fixed bed, than cells in suspension without carriers orwithout microcarriers, but nevertheless he will easily see the advantagein the oxygenation of the culture and in the nutrition thereof of such aflow direction within the culture zone. Indeed it is well known that thedesign of culture vessels is a key step in cell culture. The design ofthese must be such that there are no dead areas, not supplied with freshmedium, or in which the cells would accumulate; it is also veryadvantageous for the cells to be in direct contact with the culturemedium rather than with one another for their reproduction and/orproduction metabolism.

Hence the invention provides a flexible device adapted to suspensioncultures and cultures on carriers or on microcarriers which allows areduction in the stresses applied to the cells and which prevents theaccumulation of cells in a particular zone of the culture zone, byreducing the effect of gravity which is exerted on the cells andpreventing the presence of dead zones not supplied with fresh medium orin which the cells can accumulate. In addition, for application insuspension culture, devices according to the invention allows a gooddispersion of cells by virtue of on one hand the upward flow and on theother hand the gravity which is still partly applied to the cells.

The terms “bottom wall provided with orifices for the passage of amedium essentially free from cells” must be taken to mean a wallsituated in the bottom part of the culture zone, which may be situatedat the bottom of the culture zone or in the lower part of a verticalwall delimiting said culture zone and which allows the culture medium topass and not the cells on microcarriers or carriers or even insuspension. Similarly, the top wall provided with orifices for thepassage of a medium essentially free from cells may be a wall situatedat the top of the culture zone or at a top part of the vertical walldelimiting the culture zone with characteristics identical to thosedescribed above.

Naturally the medium which overflows from the first zone into the thirdzone can overflow over the top of the walls of the first zone and inthis way reach the third zone or can overflow via an orifice or tubeinstalled in the top or bottom part of the first medium transfer zone.The same applies to the medium which overflows from the second zone intothe fourth zone.

Advantageously, the culture medium circulation means consist of acentrifugal pump situated in a bottom part of said culture device,comprising at least one magnetic device rotating about a substantiallycentral rotation axis (real or virtual), at least one inlet and at leastone outlet for the culture medium, said circulation means being providedfor sucking the medium in a siphon created by a rotation of the magneticdevice and for propelling the medium towards the culture mediumdischarge placed in a zone external to said magnetic device and in thatsaid centrifugal pump is driven by a rotary magnetic motor provided foreffecting a circulation of medium with no communication with the outsideof the device, and at least one guidance device, designed to guide theculture medium propelled through said outlet towards the top of thevessel.

One drawback of the existing conventional bioreactor is that they areprovided with a blade or screw agitation system for providinghomogenisation of the culture medium within the culture vessel withoutnecessarily providing adequate circulation. In this case, the bioreactorcomprises a spindle provided with double fragile mechanical linings, forexample made from expensive silicon carbide and which passes through thecover of the bioreactor. This passage through the cover of thebioreactor is a serious risk of contamination and a significant risk ofbreakdown.

Other types of bioreactor comprise an external circulation of theculture medium. The medium passes through a pipe through a peristalticpump or a similar system. Obviously, this solution, although partlypreventing direct contaminations, has another drawback. It is notapplicable for long-life cultures. Indeed, the pipes used in this typeof pump are subject to wear during long life cultures, which alsoinvolves problems of sealing and contamination.

The medium circulation as described above also gives rise to a technicaldifficulty of achieving a “fountain” effect. It therefore does notsuffice to make the liquid flow through the culture zone at a very lowpressure drop, it is also necessary to maintain a difference in levelinside the culture vessel simultaneously. Indeed, bringing the liquidtowards the top of the first medium transfer zone is a key step, butalso ensuring that the medium does not accumulate excessively in thebottom of the fourth zone too. If the culture medium accumulates in thebottom of the fourth culture zone, the overflow effect will be reducedand the cyclic circulation of the medium from the first medium transferzone to the third medium transfer zone and then to the second culturezone and finally into the fourth medium transfer zone will not beoptimum.

Consequently, the circulation means according to the invention willcomprise a magnetic device rotating about a substantially centralrotation axis, and a culture medium inlet and outlet.

For small-scale cultures, the magnetic device will for example be asimple magnetic bar, driven by an external magnet, affording flow ratesof 0.6 to 6 l/min (that is to say 1 to 10 ml/sec). Because of this,there is no risk of contamination since the medium circulation systemdoes not communicate with the outside but is driven by a device externalto the culture vessel. A peristaltic pump can also be envisagedaccording to the invention, but preferentially for short durationcultures.

For cultures on a larger scale, the magnetic device will be a magnetisedrotor with a flow rate of between for example 10 and 2001/min, inparticular 20 to 1501/min and preferably 25 to 1001/min.

In a particular embodiment, a device according to the inventioncomprises a series of modules, each module comprising said first zone,said second zone, said third zone and said fourth zone, and in which theadjacent modules in said series of modules are in medium communication,said first zone and said fourth zone of each module being incommunication with said circulation means, directly or indirectly.

This embodiment makes it possible to obtain a particularly flexibledevice and allows an increase in scale up to a volume of 100 liters.

Generally, the scaling up is a complex step in the production ofrecombinant products, viruses, cell metabolites or others or in theculture of cells since this scaling up generally gives rise to problemswith zones supplied in a mediocre manner, whether it be with oxygen orwith nutriments. In the embodiment described above, the culture devicecomprises a series of adjacent modules, for example stacked orjuxtaposed. From the fourth zone of the first culture module, theculture medium reaches the medium circulation means by passing throughthe other culture modules. The invention envisages various types ofmodule of predetermined volume. For example, modules of 500 ml or 5liters, having a volume from 500 ml to 5 liters, including all volumescomprised within the range or framing the range. Consequently, for aculture of 3 liters, 6 modules of 500 ml will be used. For example, sixmodules will be stacked either in a sufficiently large vessel or bymeans of modules that can be interconnected so as to form said vessel.

In the case of the reactor of the document U.S. Pat. No. 5,501,971, if aculture volume of 3 liters is required, a culture zone height H would beneeded. The medium which reaches the bottom of the culture zone in thispatent is practically exhausted, in particular with regard to oxygen.Moreover, the authors of the U.S. Pat. No. 5,501,971 contemplate placinga oxygen sensor at the top of the culture zone and at the bottom of thelatter and oversupplies the environment of the reactor with oxygen.Unfortunately, this type of over-oxygenation is absolutely not to berecommended since this produces an oxidation of cellular components ofthe cells at the top of the culture zone and cell death follows. Inaddition, when the authors of the U.S. Pat. No. 5,501,971 contemplate ascaling up, it is in terms of width that this is carried out since interms of height it is no longer possible. Naturally, the space occupiedon the floor quickly becomes uncontrollable. As it is known for theskilled in the art, floor space is a critical parameter, especially inwhite rooms where efficiencies are calculated according to the volume ofair to be treated in order to obtain sterile air, this type of airtreatment being very expensive. The volume of air to be treated isobtained by multiplying by the height of the “white room” the surface onthe floor occupied by the bioreactor, its equipment, and personshandling the culture device.

Consequently, through the use of stacked or juxtaposed modules,according to yields to be achieved, the floor space is reduced. Inaddition, the volume of culture through which the culture medium mustpercolate before once again being in contact with the ambient air of thebioreactor is appreciably reduced. In the case of our previous example,the volume is divided by 6.

According to one advantageous embodiment, provision is therefore alsomade to design an autoclavable, empty, culture vessel with apredetermined height (made from glass or stainless steel) or an emptydisposable culture vessel, comprising the medium circulation means inthe bottom of the vessel. It will then suffice to position the number Nof modules required in the vessel above the medium circulation means andto close the vessel again by means of an adapted cover. In this case,whether for a culture of 5 liters or 50 litters, the size of the vesselremains the same.

This embodiment can be particularly advantageous for laboratories havingfew means since it is particularly inexpensive. Indeed, if the vesselsupplied is designed for a culture of 35 liters and a culture of only 5liters or 10 liters is required, it suffices to place, for example abovethe culture medium circulation means, one or two culture modules of 5liters. This particularly reduces the amount of investment since theexpensive part always remains the same for any culture from 5 to 35liters.

In addition, whether for a culture of 5 liters or 35 liters or evenmore, the floor surface will be the same, and the scaling up does notinvolve any greater volume of air to be treated. In addition, throughthe fact that the medium is, between each passage in a module, incontact with the ambient air in the reactor, the problems of scaling upare greatly reduced.

As mentioned previously, the medium circulation means are particularlyeffective by covering ranges of values particularly adapted to devicesaccording to the invention. Consequently, the flow rate in each moduleis identical and, at the level that is output, the increase in scale isalso not a problem according to the invention.

In a particularly advantageous embodiment, the circulation means areconfined in a base module, said base module being in mediumcommunication with at least one first medium transfer zone and at leastone fourth medium transfer zone, directly or indirectly.

Since the medium circulation means are often zones presenting risk ofcross contamination or external contamination through the presence ofsome not accessible zones for cleaning, procuring a base module whichcan for example, without however being limited thereto, be inserted in asimple glass vessel is particularly advantageous. This base module isadapted to the culture modules, allowing the communication of mediumbetween the first medium transfer area of all the modules present andthe medium outlet of the medium circulation means and allowing mediumcommunication between all the fourth zones of all the culture modulespresent and the inlet of culture medium into the medium circulationmeans, directly or indirectly, that is to say passing through anothermodule or not.

Devices preferably also comprises a head module, said head modulecomprising at least the cover. This head module being able to bedesigned to close off the superimposition mentioned above.

In an advantageous embodiment, at least one fourth zone comprises atleast one substantially vertical or inclined flow wall.

The presence of this flow wall reduces the formation of foam which couldappear during the overflow from the second culture zone to the fourthmedium transfer zone. Indeed, without this flow wall, the flow of mediumfrom the second to the fourth zone would be a turbulent flow, whichwould necessarily have as its consequence an undesirable formation offoam. It should be stated that the formation of foam is a major problemin many culture methods since the culture medium is rich in proteins.Stirring a fluid rich in protein always causes the appearance of foam.Consequently a turbulent flow would have this same consequence, and thisis why, advantageously, the invention comprises said flow wall forreducing the flow turbulence.

In addition, this fourth medium transfer zone is also a zone in whichthe medium is in contact with the ambient atmosphere of devicesaccording to the invention. The presence of the flow wall improves thiscontact and therefore the exchanges of oxygen between the ambientatmosphere and the culture medium, by increasing the gas-liquid contactsurface area.

Advantageously, in order to stabilise the film, it is also possible toadd additives to the culture medium in order to modify the rheologicalproperties of the water such as the additives included in the groupconsisting of surfactants, Pluronic F68, glycerine, quaternary ammoniumsand any other additive for modifying the rheological properties of theculture medium.

In a particularly preferred embodiment, the essentially vertical orinclined flow wall comprises a hydrophilic membrane.

Indeed, if the flow wall is not or does not comprise a hydrophilicsurface, it may be very difficult to obtain a film of medium on thissurface. In addition, the film, when it is formed on a conventionalwall, is unstable. Consequently, by covering the flow wall with thehydrophilic membrane which fulfils the role of a sponge, the medium isnaturally spread and flows evenly. Consequently, the contact surfacebetween the film of medium and the ambient atmosphere is greatlyimproved, which permits oxygenation compatible with high cell densities.The coefficients of total transfer of oxygen obtained are from about10-³ to about 10-² s⁻¹.

If, in the embodiment with culture modules, some or all of the fourthzones comprise a flow wall with a hydrophilic membrane, the gas-liquidexchanges are further improved. Consequently, even the last module inthe series is supplied with an oxygenated medium. In some cases of theprior art with circulation of medium, when a culture is produced, forexample with a volume of 200 liters, the cells at the end of the mediumcirculation are relatively undersupplied and the cell growth is nothomogenous in the bioreactor.

In devices according to the invention, this is not the case; noundersupplied or dead zone exists.

In one advantageous embodiment, devices according to the inventioncomprise at least one gas inlet orifice and one gas outlet orifice.

The culture vessel preferably comprises at least one gas inlet orificeand one gas outlet orifice. In this way, it is possible to enrich theambient atmosphere of devices according to the invention with oxygen forexample, as the oxygen is consumed by the cells. It is also possible tosupply the ambient atmosphere with other gases, for example by addingCO2 in order to modify the pH, or any other gas generally used in cellculture. The outlet orifice making it possible to prevent overpressuresand to discharge the gas with a low oxygen content or simply part of thegas of the ambient atmosphere in order to reduce the ambient pressure ofa device according to the invention. Also, provision is made to be ableto close off or “strangle” this gas outlet in the case where a slightoverpressure is desirable.

The gas inlet can be situated or connected to the first, to the second,to the third or to the fourth zone of the culture vessel. The first andfourth being preferential zones. The third medium transfer zone is notvery accessible for gas-liquid contacts and the second zone of theculture vessel is the cell culture vessel, in which direct gas-liquidcontact could present a risk of oxidation of the cell components, whichis not desirable.

Advantageously, the gas inlet orifice is connected to at least onefourth medium transfer zone.

Because the fourth medium transfer zone also serves as an oxygenationzone since it is in the latter that the gas-liquid contacts are thegreatest, with or without a flow wall, it is advantageous for it to bethe latter that directly receives the addition of fresh gas in order topromote the oxygenation of the medium. In addition, the culture mediumwhich overflows into this fourth zone is the medium used up by thecells, and therefore part of the oxygen is consumed, it may also need amodification to the pH and it is therefore an advantage for the gas(oxygen, air, CO2 or other) to enter through this fourth culture zone inorder to increase the efficiency of any regulation or oxygenation ormodification of pH.

In a particular embodiment, the gas inlet is connected to a spargertube.

The invention, in a particular embodiment, provides for a dispersion ofgaseous bubbles to be effected within a device according to theinvention. The dispersion of bubbles can be effected by a spargerimmersed in the culture medium in the first, second, third and fourthmedium transfer zone (large bubbles or microbubbles, according to theapplication). The dispersion of bubbles will preferably be effected by asparger immersed in the culture medium in the first or fourth mediumtransfer zone.

In a particular embodiment, the cover of the culture vessel is connectedto at least part of said top wall of at least one second culture zone.

This particularly preferred embodiment allows a simplified taking ofsamples which minimises the risks of contamination. Particularly duringcultures on microcarriers, taking samples is not generally a stepwithout any risk. To measure the cell density, at the present time thereexist only a few means, which are also not very reliable and tedious.Consequently the best means of measuring the cell density is samplingcarriers and observing them under the microscope, possibly aftercolouring.

Indeed, in conventional bioreactors, and in particular in the bioreactorof the U.S. Pat. No. 5,501,971, taking samples is either impossiblebecause the top wall of the culture zone cannot be opened simply or itrepresents a serious risk of contamination. Indeed, the user should openthe cover of the culture vessel, which is often bulky and heavy andtherefore difficult to move in a sterile flow, and then hold the cover,generally fixed to a blade or other device for raising the medium andtherefore heavy and bulky, and he should ensure that he does not touchanything and hold it in one hand in order not to contaminate it. Next,with the other hand, he must open the top wall of the culture zone andhold it in the other hand. Then, by means of a clamp in a third hand, hemust sample one or more carriers in order to be able to evaluate thecell density. This requires the presence of a second user or impressivedexterity.

The invention greatly simplifies this step of taking samples byprocuring a cover fixed to the top wall of the culture zone; all thatneeds to be done then is to slightly lift the cover, the top wall of theculture zone rising simultaneously, and to introduce a sterile clamp ora sampling tool such as a pipette or the like for sampling one or morecarriers in order to evaluate the cell density. The risks of touching anon-sterile object and the risks of contamination are greatly reduced.

Advantageously, in certain embodiments, devices according to theinvention also comprise a heating means, designed to heat thetransferred culture medium. This heating means can advantageously besituated in the fourth medium transfer area or at the medium circulationmeans. Naturally, the first zone can also comprise this heating means.

The heating means can be an electrical element, an electrical coil orany other heating means generally used in the field of cell culture,such as for example a thermostatically controlled double jacket.

Indeed, during large or very large scale culture, it is not always easyto place a device according to the invention in a thermostaticallycontrolled device or room. Consequently the invention solves thisproblem by directly placing heating means in order to control theculture medium thermostatically and to afford an even temperaturethroughout the culture vessel. Preferably, a device according to theinvention provides a heating of the medium without any overheatingpoint.

In certain embodiments, devices according to the invention comprisessensors for measuring culture parameters, said sensors being in contactwith the culture medium. Culture parameters means, amongst other things,the dissolved oxygen partial pressure, the pH, the temperature, theoptical density, certain concentrations of nutriments, such as lactate,ammonium, carbonates, glucose or any metabolic product or product to bemetabolised which could for example reflect the cell density.

It can also be contemplated according to the invention using regulationloops according to these parameters. These regulation loops would forexample modulate the quantity of oxygen to be injected into the gaseousatmosphere according to the value of the dissolved oxygen partialpressure present or the quantity of dissolved oxygen consumed by thecells. It could inject CO₂ according to the pH value obtained by thesensors or any other type of regulation generally used in this type ofculture.

The sensors are preferably arranged in a bottom part of at least onefourth zone. The fourth medium transfer zone is a preferential zone forthe positioning of the sensors since the values obtained by the latterare clearly representative of the cell consumption since this zone hasthe medium coming from the culture zone passing through it.

Advantageously, said sensors are disposable optical sensors, providedfor transmitting an optical signal representing the parameters to bemeasured through said culture vessel to an optical signal receiver,external to said device.

In a particularly preferential embodiment, the device comprises a seriesof modules, each culture module comprises in its top part a first fixingmeans and in its bottom part a second fixing means, in which said basemodule also comprises in a top part a first fixing means and said headmodule also comprises in its bottom part a second fixing means, saidfirst fixing means and said second fixing means being complementaryfixing means for producing a stacking sequence from the bottom to thetop of a base module, at least one culture module and a head module.

Advantageously, said first and said second fixing means comprise meansfor producing said stacking sequence in a gas- and liquid tight manner.

Indeed, according to the invention, it is possible to design a series ofmodules where the wall of each module would constitute part of the wallof the culture vessel. Next, it suffices to assemble this series ofculture modules with a base module comprising the circulation means anda head module comprising the cover. The assembly formed by these moduleswould then constitute the culture device. According to the culturevolume necessary, a number N of modules would be fitted together toconstruct one's own culture device with a height H.

In a very particular embodiment, devices according to the invention is adisposable device.

There exist at the present time a quantity of bioreactors which giveexcellent cell culture results on a small and large scale.Unfortunately, these bioreactors are expensive in terms of cleaning,sterilisation, labour, location and space occupied. Indeed, inparticular for the production of clinical batches or products ofpharmaceutical interest, it is essential for the bioreactors to beplaced in sterile white rooms. A 500 liter bioreactor occupies more thantwenty times its volume in a white room. The white room criterion is thevolume used by an installation, that is to say, if the installationoccupies 1 m₂ of floor surface, the volume of the air to be treated willbe (1 m₂ plus the surface area necessary for the user) multiplied by theheight of the white room since the volume of air above the floor surfaceis also to be treated. In addition, the sterilisation, asepticisation,washing, sanitisation, etc protocols, which are steps required both fora bioreactor and for the room occupied, are extensive and tediousprotocols which impose enormous costs both with regard to labour andinstallation of products. This is why, principally in pharmaceutical,biological and biochemical laboratories and in white rooms, disposableequipment, generally less bulky, not requiring cleaning, sanitisation,sterilisation and asepticisation, is being increasingly used each day.

Alternative solutions to reusable conventional bioreactors exist, forexample culture is known in a disposable sterile container which isstirred by a stirring plate reproducing a wave movement, for example theWAVE® bioreactor. Unfortunately, such a bioreactor presents a problem ofscaling up since a 500 liter container, in so far as the stirring platecan be sized, has an enormous floor surface which has a white room airtreatment cost that it is impossible to assume, without mentioning thedifficulty of handling such containers, taking samples and placingculture parameters sensors.

Other solutions exist, such as stirred disposable flasks called“spinners”. The scaling up of these flasks is also impossible and theflasks have a low oxygen transfer as well as stresses on the cellsduring stirring.

There also exists culture in a CELLCU BE® or CELLFACTORY® system. Such asystem is difficult to regulate and is bulky. In addition, the oxygentransfers are poor and it requires incubators of large size. Once again,scaling up is tedious.

The BELLOCELL® system is also known. This system is based onimmobilising cells in porous matrices, which are packed in a culturezone. The medium is in a lower zone provided with a compressiblebellows. The medium rises and falls alternately in order to immerse thematrices in the medium and then expose them to the ambient air.

Unfortunately, the scaling up of this system is also difficult. It isdifficult to regulate and measure the culture parameters. In addition,the cells undergo tension stresses on their surface, being exposed firstof all to a falling medium edge, drying, and then a rising medium edge,which is detrimental to their growth.

In summary, there exists at the present time no disposable cell culturesystem adapted for culture on a large and small scale which is easy touse, both in a white room and in the laboratory.

The invention therefore procures a very innovative solution which solvesa major part of these drawbacks by procuring an unexpected system,applicable on a small and large scale, suitable for culture insuspension, on carriers or on microcarriers, the stirring of whichaffords homogeneity of the culture medium without dead zones nor cellaccumulation zones. In addition, the risks of contamination because ofthe absence of a central spindle are particularly low, or evennon-existing.

In fact, devices according to the invention present all the advantagesof conventional bioreactors, as mentioned here previously, whilst beingdisposable. Devices according to the invention is stirred magneticallyby medium circulation means based on a centrifugal pump, which do nothave any contact with the outside. Heating means, for example anelectrical coil, also afford homogeneous heating of the medium withouthaving contact with the outside.

In addition, as mentioned above, in a particularly preferential manner,the culture device according to the invention comprises sensors whichare disposable optical sensors, designed to transmit an optical signalrepresenting the parameter to be measured through said culture vessel toan optical signal receiver, external to said device.

Consequently, the culture parameters are also measured through a wall ofthe device and this does not involve any contact with the outside,unlike the dissolved oxygen or pH probes passing through the cover ofclassical bioreactors, presenting a risk of poor cleaning in these probepassage orifices and of contamination through lack of a seal.

The sensors can be situated in the bottom part of the device or in thetop part or both. When the sensors are present in the top and bottomparts, this makes it possible, by a simple mathematical differenceoperation, related or not to the number of cells, to make a continuousmeasurement of the cell respiration.

Other embodiments of the device are described further below.

Another object of the invention is the use of the culture deviceaccording to the invention for cell culture in suspension onmicrocarriers or on carriers. Indeed, when devices according to theinvention are used in culture in carriers or microcarriers, the carriersor microcarriers are confined in said second culture zone. When thedevice is used for suspension cell culture, and therefore withoutcarriers or microcarriers, the wall provided with an orifice is amembrane permeable to the medium but not permeable to the cells. Thesize of the pores of this membrane is a function of the all size. Theinvention also relates to the use of the culture device according to theinvention for producing recombinant products, viruses, metabolites andthe like.

Recombinant products means proteins of interest for research in thepharmaceutical sector, therapeutic or prophylactic molecules,antibodies, plasmids or any other molecule able to be produced by cellsin culture, whether this be production by secretion or intracellularproduction.

The invention also relates to a method of culturing cells in a culturevessel with circulation of the culture medium, comprising:

introduction of culture medium into culture medium circulation means,

discharge of culture medium from said culture medium circulation means,

at least one first transfer of culture medium into a first culturemedium upward transfer zone,

at least one second transfer of culture medium into a second cellculture zone.

This method is characterised in that it also comprises:

at least one third culture medium transfer subsequent to said firstculture medium transfer into a third culture medium transfer zone, byoverflowing from the first culture medium transfer zone to the thirdculture medium transfer zone,

at least one fourth culture medium transfer subsequent to said secondculture medium transfer into a fourth culture medium transfer zone, byoverflowing from the second culture zone into the fourth culture mediumtransfer zone,

and in that said second culture medium transfer is a downward culturemedium transfer.

As mentioned above, unlike the culture method of the U.S. Pat. No.5,501,971, the culture medium travels through the culture zone upwards,which prevents the accumulation of cells in the bottom of the culturezone and reduces the pressure forces on the cells. The culture methodaccording to the invention is therefore a particularly innovative methodwhich allows culturing without a dead zone and without any place wherecells accumulate whilst permitting culturing at very high efficiency.

Advantageously, the method also comprises an oxygenation of the culturemedium during one or more of said transfers.

Said oxygenation preferably occurs through a direct gas-liquid contactduring one or more of said transfers.

In a particular embodiment, the oxygenation is carried out during thefourth culture medium transfer, said fourth culture medium transferbeing a flow of said culture medium along a flow wall.

In an advantageous embodiment, the method, in order to stabilise thefilm, provides for the addition of additives to the culture medium inorder to modify the rheological properties of the water such as theadditives included in the group consisting of surfactants, Pluronic F68,glycerine, quaternary ammoniums and any other additive for modifying therheological properties of the culture medium.

Also advantageously, the flow of the culture medium is a flow along ahydrophilic wall.

Other embodiments of the method according to the invention are indicatedin the accompanying claims.

Other characteristics, details and advantages of the invention willappear more clearly in the light of the following description, ofparticular non-limiting embodiment of the invention, while referring tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an outline diagram of a culture device according to anembodiment of the invention.

FIG. 2 is an outline diagram of a variant of the medium circulationmeans.

FIG. 3 is an outline diagram of a culture device according to theinvention comprising several successive culture zones, particularlyadapted for a scaling up.

FIG. 4 illustrates highly schematically a variant of FIG. 3.

FIG. 5 is a transversal section of a bottom part of the base moduleconfining the medium circulation means of a preferred embodiment ofdevices according to the invention.

FIG. 6 is a top view of the bottom part of the base module confining themedium circulation means as illustrated in FIG. 5.

FIG. 7 is a transversal section of a top part of the base moduleconfining the circulation means of the same preferred embodiment ofdevices according to the invention.

FIG. 8 is a top view of the top part of the base module confining thecirculation means of a device as illustrated in FIG. 7.

FIG. 9 is a transversal section of the culture device according to theinvention produced by an assembly of modules, in particular disposablemodules.

FIG. 10 is an exploded view of FIG. 9.

FIG. 11 is outline diagram of a culture device according to anembodiment of the invention, showing a solid element (35) within thesecond zone (5).

FIGS. 12 and 13 are outline diagrams of a culture device according to anembodiment of the invention, showing a second zone (5) which does notcomprise internal to the second zone (5) portions of a first zone (3) orthird zone (4).

FIG. 12 shows a particular embodiment with a closed element 36 bpositioned above opening 36 a.

FIG. 13 shows a particular embodiment with a flow regulating element 36c positioned above opening 36 a.

In the drawings, a same or similar reference sign has been allocated toa same or analogous element.

As can be seen in FIG. 1, the culture device 1 comprises a substantiallyvertical and cylindrical culture vessel 2, although other forms can alsobe envisaged according to the invention, for example any prismaticshape, preferably regular. The culture vessel comprises at least fourzones in communication with one another. From the centre of the vesseltowards the outside, the vessel comprises a first zone 3, a third zone 4a, 4 b, a second zone 5 and a fourth zone 6.

The zone comprising cells has been hatched and comprises the letter Cwhilst the medium is shown by the letter M.

The culture vessel 2 comprises medium circulation means in its bottompart. The medium circulation means are, in this preferential embodiment,composed of a magnetic device 7, for example a magnetic bar 7 inrotation about a central rotation axis 8, real or virtual, a first endof which is housed in a top engagement means 9 and a second end of whichis housed in a bottom engagement means 10. The magnetic bar is driven bya rotary magnetic drive motor external to the culture vessel 2 and whichis not shown here. The circulation means comprise at least one mediuminlet 11. The medium inlet 11 comprises at least one first end whichends in a diversion baffle 12 for the flow of medium. The magnetic barfunctions as a centrifugal pump, that is to say the medium is suckedinto a relatively central zone by the movement of the medium created bythe bar and the medium is propelled outwards with respect to the centralpoint. The medium diversion baffle 12 guides the medium in therelatively central zone of the bar so that the medium is sucked thereinand is then propelled outwards. Advantageously, the inlets are in thesame plane (star configuration) and the number of inlets 11 will be anumber such that their positions will exhibit symmetry. Moreparticularly, if three inlets are considered, it is advantageous forthem each to be separated from one another by an angle of approximately120°, if the number of inlets equal 4, the inlets will be separated fromone another by an angle substantially equivalent to 90°, if the numberof inlets is equal to 10, the inlets will be disposed with a separationangle approximately equal to 36°.

The medium circulation means also comprises at least one medium outlet13. The medium outlet 13 is advantageously situated at the point wherethe medium is propelled by the centrifuge effect of the magnetic bar.

Advantageously, the number of outlets 13 will be a number such thattheir positions will exhibit symmetry. More particularly, if threeoutlets are considered it is advantageous for them each to be separatedfrom the other by an angle of approximately 120°, if the number ofoutlets is equal to four, the outlets will be separated from one anotherby an angle substantially equivalent to 90°, if the number of outlets isequal to 10, the outlets will be disposed with a separation angle ofapproximately 36°.

Preferably, the outlets are not situated in the same horizontal plane asthe inlets. The bottom part of the culture vessel comprises at least onemedium guiding means 14, adjacent to said at least one outlet 13, whichguides the culture medium propelled towards to the top of the culturevessel 2.

The first zone 3 of the culture vessel 2 is a substantially central zoneand is a medium transfer zone. The first zone 3 comprises a basal part 3a and in particular embodiments optionally also a cylindrical part 3 b.The diameter of the basal part 3 a is less than the diameter of theculture vessel 2. The basal part 3 a is in medium communication withsaid at least one medium outlet 13 of the medium circulation means. Thebasal part 3 a is reduced in the top part of the first zone 3 to acylinder 3 b with a smaller diameter than the basal part 3 a. The topcylindrical part 3 b comprises an external wall and is in direct mediumcommunication with the basal part 3 a of said first medium transferzone.

The third zone 4 is a medium transfer zone, external to the first mediumtransfer zone 3. The third zone also comprises a substantially basalpart 4 a (in the form of a sleeve) and in particular embodimentsoptionally also a substantially cylindrical top part 4 b.

The substantially cylindrical part 4 b of the third medium transfer zone4 is essentially concentric with the substantially cylindrical part 3 bof the first medium transfer zone 3 and these two parts are in mediumcommunication. The medium communication is achieved by means of anorifice or a tube, by overflowing (as shown in the figure) via overflow(37) or any other possible means for achieving this communication. Thesecond zone 5 is a cell culture zone, with or without carriers ormicrocarriers.

The second zone 5 is also in the form of a sleeve, at the centre ofwhich are the first and third medium transfer zones 3 and 4.

The second zone 5 comprises a bottom wall 15 and a top wall 16, eachwall 15 and 16 being provided with orifices 17 allowing a transfer ofcultured medium essentially free from cells. The second culture zone 5is in medium communication with the relatively basal part 4 a of thethird medium transfer zone 4 by means of orifices 17 in the bottom wall15 allowing the medium to pass.

The fourth zone 6 is a medium transfer zone, external to the secondculture zone 5 but internal to the culture vessel 2. The fourth zone 6is in medium communication with the second culture zone 5. It is also inmedium communication with the medium circulation means, via said atleast one inlet 11. The medium communication is achieved by means of anorifice or a tube, by overflowing or by any other possible means forachieving this communication.

The particular embodiment described here comprises a substantiallycylindrical culture vessel, but other embodiments can also be envisaged,as mentioned previously, for example a substantially prismatic vessel,preferably regular. Obviously, this is also the case with the variousmedium and culture transfer zones. They can also be prismatic,preferably regular, any combination of shapes being possible. In thiscase, the term sleeve must be envisaged as an envelope with across-section similar to the cross-section of the prism envisaged.

When the medium circulation means are in operation, the medium leavesthem through said at least one outlet 13, when there are several ofthem, through the various outlets 13, and is diverted by the guidingmeans 14, it ends up in the substantially basal part 3 a of the firstmedium transfer zone 3. The structure of the first medium transfer zone3 and the output of the pump require the medium to be directed towardsthe substantially cylindrical part 3 b of the first medium transfer zone3. When it reaches the top of the wall of the substantially cylindricalpart 3 b, it overflows via overflow (37) into the third medium transferzone 4.

The direction of circulation of the medium M is shown by the arrows inthe figures.

It is clear to the skilled in the art that, in this particularembodiment, the wall of the substantially cylindrical part 3 b of thefirst medium transfer zone 3 is less high than the wall of the thirdmedium transfer zone 4 for reasons of efficiency and flow rate, but hewill easily understand that the wall of the substantially cylindricalpart 3 b of the first medium transfer zone 3 can also be higher than thewall of the substantially cylindrical part 4 b of the third mediumtransfer zone 4.

The medium is therefore subjected to the flow rate imposed by the pumpand to gravity, it is directed downwards from the third medium transferzone 4 running down the substantially cylindrical part 4 a and reachesthe substantially basal part 4 b of the third medium transfer zone 4.Next the flow of medium has a rising direction through a communicatingvessels effect by the imposed flow rate of the pump and reaches the topof the second culture zone 5. The medium reaches the second culture zone5 from the third medium transfer zone 4 via the orifices for the passageof medium substantially free from cells 17 in the bottom wall 15 of thesecond culture zone 5.

As already mentioned above, the medium passage orifices 17 are sizedaccording to the type of culture. If the culture is a culture withoutcarrier, the wall 15 or 16 comprising orifices 17 will be a porousmembrane where the pore size is less than the diameter of the cells. Ifthe culture is on microcarriers or on carriers, the size of the orifices17 will be less than the size of the microcarriers or carriers.

When the medium flow edge reaches the top of the wall of the secondculture zone 5, it overflows into the fourth medium transfer zone 6.Naturally, if orifices are present or a tube, it must be understoodthat, when the medium flow edge reaches the orifice or tube, it flowsinto the fourth zone 6.

In the particularly preferential embodiment of the invention, the fourthmedium transfer zone 6 comprises an inclined wall 18 on which the mediumflows when it passes from the second zone 5 to the fourth zone 6. Theinclined wall preferably comprises a hydrophilic membrane in order toimprove the formation of the film on said inclined wall 18. The filmmust preferably be laminar in order to prevent as far as possible theformation of foam. In order to stabilise the film, it is also possibleto add additives to the culture medium in order to modify therheological properties of the water, in particularly of the culturemedium, such as the additives included in the group consisting ofsurfactants, Pluronic F68, glycerine, quaternary ammoniums and any otheradditive for modifying the rheological properties of the culture medium.

The hydrophilic membrane will for example be a membrane consisting ofpolyoxyethylene.

The formation of the film on the inclined wall is an important stepsince it allows oxygenation on “thin film”. Indeed the gaseous volumewith respect to the quantity of medium in this fourth medium transferzone is large and improves exchanges. In addition, the formation of thefilm on an inclined wall increases the gas-liquid contact surface area.

As can be seen in FIG. 1, the culture vessel preferably comprises acover 19 through which at least one gas inlet orifice 20 and at leastgas outlet orifice 21 pass. The gas inlet orifice 20 is preferablysituated so as to communicate directly with the fourth medium transferzone 6. In some variants, it may be preferable for the gas inlet orifice20 to be present on the vertical wall of the culture vessel 2 or at thebottom of the culture vessel 2, that is to say the gas passes by meansof an orifice 20 through the wall of the culture vessel 2 opposite tothe cover 19, and for this orifice 20 to be provided with a tube inorder to end above the liquid level (see FIG. 9).

In this embodiment, the cover 19 is fixed by fixing means 22 to the topwall 16 of the second culture zone 5. In variants, the cover 19 can bemade an integral part of the top wall 16 of the second culture zone 5,this part opening when the cover 19 of the culture vessel 2 is raised.In this way, it is easy to take off a cell sample with or withoutcarriers in order for example to evaluate the cell density, thestructure of the cells and other physical characteristics of the cellwhich reflect the health of the culture. Indeed, connecting the twotogether makes it possible to open the culture compartment 5 simply byraising the cover 19 of the culture vessel 2.

In the case of culture in suspension, it could be advantageous toconnect a porous membrane to the top wall 16 provided with orifices 17of the second culture zone 5, this assembly can improve the rigidity ofthe cover/membrane assembly for taking samples.

FIG. 2 illustrates a variant of the magnetic device of the mediumcirculation means. Here the magnetic bar 7 has the shape of a helix. Thedesign of the magnetic device 7 with a substantially central rotationaxis 8 will depend essentially on the volume of the culture. Indeed, forsmall cultures, the invention sets out to be able to us a simple barsuch as a magnetic chip for circulating the medium. For large volumes,the invention envisages a magnetic rotor, also driven by an externalmotor, for example rotors like the ones used in aquariums which allowhigh medium circulation rates.

It can also, according to the invention, be envisaged using bubbleproduction devices (not shown), more commonly referred to as “spargers”or “microspargers” according to the size of bubble produced.

Advantageously, when bubbles will be used, the pierced end of the bubbleproduction device, for example of the tube, will be immersed in themedium at the bottom of the fourth medium transfer zone or in the firstmedium transfer zone. When this type of oxygenation is chosen, it isalways also possible to continue the oxygenation on thin film, whichmakes it possible to reduce the flow of gas and to form fewer bubblesand therefore to reduce the formation of foam. In this case, provisionis also made for having two gas inlets in the cover of the culturevessel or on the vertical wall of the latter. In addition, it is alsopossible to envisage that the bubble production device be present solelyas an SOS procedure, and used solely when necessary.

The culture device also comprises a series of culture parameter sensors23, for example for the dissolved oxygen partial pressure pO2, aciditypH, temperature, cloudiness, optical density, glucose, CO2, lactate,ammonium and any other parameter normally used for monitoring cellcultures. These sensors are preferably optical sensors which do notrequire connections between the inside of the culture vessel and theoutside thereof. The preferential position of these sensors 23 is acritical position in that it is advantageous for these to be situatedclose to the wall of the culture vessel 2, for them to be in contactwith the medium M and preferably in strategic positions, as in the zonethrough which the medium M passes before it passes through the cells orjust after.

In fact, the invention contemplates particularly procuring a disposablebioreactor for all the reasons of simplicity and economy mentionedpreviously. Consequently, this is why the connections between the insideand the outside of the culture vessel have been reduced. In addition,the bioreactor according to the invention also envisages procuring aparticularly reliable bioreactor in which the risks of contamination areparticularly low by being disposable.

As can be seen in FIG. 3, an embodiment of a device according to theinvention also envisages a modular design which comprises a series ofmodules for cultures on a larger volume. For example, with this type ofmodular design, culture volumes of around 500 ml to 100 liters are forexample envisaged, through the use of a very limited number of standardmodules.

According to the invention it is envisaged providing a series of modulesthat can be “slipped” around the first medium transfer zone 3 to beplaced in a standard culture vessel 2 comprising medium circulationmeans and a cover 19.

In a particularly flexible variant, the invention sets out to procure amounting system which comprises various standard modules. These standardmodules are for example a circulation means module to be placed at thebottom of the assembly, one or more culture modules and a cover module.According to the invention, although other means of fixing these modulescan be envisaged, the modules will be clamped on one another, forexample by means of rapid connectors perfectly impermeable from theliquid and gaseous point of view.

Consequently, according to the type of culture and the required volume,the user will be able to take from his stock a base module comprisingthe medium circulation means, he will also have to take therefrom thenumber of culture modules that he requires according to the requiredculture volume and then take a head module corresponding to the cover.Next, all these modules being packaged in sterile fashion, he willmerely need to unpack them and “clip” them one above the other. Thestacking can form the “disposable bioreactor” or can be placed in anappropriate vessel.

FIG. 3 therefore illustrates an embodiment of the modular culture deviceaccording to the invention.

The culture device 1 comprises a culture vessel 2 which comprises mediumcirculation means like those explained in detail in FIG. 1. The basemodule m₀ comprising the circulation means can be fixed to the bottom ofthe culture vessel 2 or can also be slid into the culture vessel 2 (theembodiment depicted) in order to be able to dispose it and to useanother one for another culture and thus prevent cross contaminations.

The base module m₀ comprises the circulation means. As in FIG. 1, thesecirculation means comprise a magnetic device 7, rotating about a centralrotation axis 8, a first end of which is housed in a top engagementmeans 9 and a second end of which is housed in a bottom engagement means10. The circulation means comprise at least one medium inlet 11. Themedium circulation means also comprise at least one medium outlet 13.The base module m₀ of the culture vessel comprises at least one mediumguiding means 14 adjacent to said at least one outlet 13, which guidesthe culture medium propelled towards the top of the culture vessel 2.

The culture vessel 2 comprises a series of culture modules (m₁, m₂, . .. , m_(n)) which are, in this embodiment, stacked one above the other.It could also be envisaged that they be simply adjacent to one another,that is to say placed side by side. In the embodiment illustrated inFIG. 3, the modules are clamped to one another by means of rapidconnectors 24 or clips.

Each culture module m₁, m₂, . . . , m_(n) comprises a first 3, a second4, a third 5 and a fourth zone 6. These zones 3, 4, 5, 6 have each thesame function as that mentioned in FIG. 1.

In addition, it may be advantageous for each module to comprise a gas orgas mixture inlet (not illustrated) in communication with the fourthzone 6 of each culture module. The vessel may also comprise for its partan outlet for the excess gas or gas mixture (not illustrated). Forexample, the gas inlet orifice may be present at the bottom of theculture vessel 2, that is to say the gas passes by means of an orificethrough the wall of the culture vessel 2 opposite to the cover 3 andthis orifice 20 is provided with a tube in order to end above liquidlevel (see FIG. 9) of the module m₁. Consequently, the gaseous mixturereaches the fourth medium transfer zone 6 of this module. The module m₂placed above the module m₁ can also comprise a tube which enables thegaseous mixture present in the fourth zone 6 of the culture module m₁ tocommunicate with the fourth zone 6 of the module m₂. This tube thereforeadvantageously passes through the bottom wall of the module m₂.

In certain embodiments, for long duration culture, it may beadvantageous to replace part of the culture medium with fresh medium orto carry out an addition of nutriment. Consequently, the base module m₀can then comprise a nutriment inlet (not illustrated). Alsoadvantageously, the culture vessel can comprise, at the mediumcirculation means, a medium outlet (not illustrated) in order to preventoverflowing.

In a similar manner, the culture vessel 2 comprises a head modulecomprising a cover 19, advantageously connected to a top wall 16provided with medium passage orifices 17 by fixing means 22 in order tosimplify taking samples in the module m_(n) situated above, as in FIG.1.

In addition, advantageously culture parameter sensors can also beprovided in each culture module. It is also possible to provide sensorsin only one or several culture modules at all zones or in the basemodule.

In the embodiment illustrated in FIG. 3, the medium circulates in thefollowing manner. To simplify the explanation, we will use only twoculture modules m₁ and m₂ and a base module m₀, but it is certain thatthe culture device according to the invention can comprise a very largenumber of them.

In the base module m₀, the medium is propelled from the mediumcirculation means M via said at least one outlet 13, when there areseveral of them, through the various outlets 13 and is diverted by theguiding means 14. It ends up in the substantially basal part 3 a of thefirst medium transfer zone 3. The substantially basal part 3 a of thisembodiment is a zone common to all the culture modules and, in theembodiment illustrated, is situated in the base module. This is validwhether the culture modules are stacked or juxtaposed.

The structure of the first medium transfer zone 3 of an embodiment of adevice according to the invention and the output of the pump require themedium to be directed towards the substantially cylindrical part 3 b ofthe first medium transfer zone 3 of the first module m₁; towards thesubstantially cylindrical part 3 b of the first medium transfer zone 3of the second module m₂.

In this embodiment, it is the assembling of the modules which creates alarge first medium transfer zone 3 comprising a substantiallycylindrical part 3 b.

When the medium reaches the top of the wall of the substantiallycylindrical part 3 b of the second culture module m₂, it overflows intothe third medium transfer zone 4 of the second culture module m₂.

The direction of circulation of the medium M is shown by arrows.

The medium is therefore subjected to the flow rate imposed by the pumpand to gravity, it is directed towards the bottom of the third mediumtransfer zone 4 of the second culture module m₂, flowing down thesubstantially cylindrical part 4 a of the second culture module m₂, andreaches the substantially basal part 4 b of the third medium transferzone of the second culture module m₂. Next, the flow of medium has arising direction through a communicating vessels effect and through theimposed flow rate of the pump and reaches the top of the second culturezone 5 of the second culture module m₂. The medium reaches the secondzone 5 of the second culture module m₂ from the third medium transferzone 4 of the second culture module m₂ via the orifices for the passagesof medium substantially free from cells 17 of the bottom wall 15 of thesecond culture module m₂.

When the medium flow edge reaches the top of the external wall of thesecond culture zone 5 of the second culture module m₂, it overflows intothe fourth medium transfer zone 6 of the second culture module m₂.Naturally, if orifices or a tube are present in this external wall ofthe culture zone 5, it is necessary to understand that, when the mediumflow edge reaches the orifice or tube, it flows into the fourth zone 6of the second culture module m₂.

In the particularly preferential embodiment of the invention, the fourthmedium transfer zone 6 of the second culture module m₂ comprises aninclined wall 18 on which the medium flows when it passes from thesecond zone 5 of the second culture module m₂ to the fourth zone 6 ofthe second culture module m₂. The inclined wall preferably comprises ahydrophilic membrane in order to improve the formation of the film onsaid inclined wall 18. The film must preferably be laminar in order toprevent as far as possible the formation of foam. In order to stabilisethe film, it is also possible to add additives to the culture medium inorder to modify the rheological properties of the water, as mentionedbefore.

Next, the culture medium present in the fourth medium transfer zone 6 ofthe second culture module m₂ overflows either through a tube or over thetop (D) of the wall of the fourth medium transfer zone 6 of the secondculture module m₂ into the third medium transfer zone 4 of the firstculture module m₁.

The medium is therefore subjected to the flow rate imposed by the pumpand to gravity, it is directed downwards from the third medium transferzone 4 of the first culture module m₁, flowing down the substantiallycylindrical part 4 a of the first culture module m₁, and reaches thesubstantially basal part 4 b of the third medium transfer zone of thefirst culture module m₁. Next, the flow of medium has an upwarddirection through a communicating vessels effect and through the flowrate imposed by the pump and reaches the top of the second culture zone5 of the first culture module m₁. The medium reaches the second zone 5of the first culture module m₁ from the third medium transfer zone 4 ofthe first culture module In₁ via the orifices for passage of mediumsubstantially free from cells 17 in the bottom wall 15 of the firstculture module m₁.

When the medium flow edge reaches the summit of the wall of the secondculture zone 5 of the first culture module m₁, it overflows into thefourth medium transfer zone 6 of the first culture module m₁. Obviously,if orifices or a tube are present in this wall, it must be understoodthat, when the medium flow edge reaches the orifice or the tube, itflows into the fourth medium transfer zone 6 of the first culture modulem₁.

The fourth medium transfer zone 6 of the first culture module m₁ canalso comprise an inclined wall 18 on which the medium flows when itpasses from the second culture zone 5 of the first culture module m₁ tothe fourth medium transfer zone 6 of the first culture module m₁. Theinclined wall is possibly provided with a hydrophilic membrane as above.

Next, the medium returns to the base module m₀ and to the mediumcirculation means through the inlet (pipe) 11, that is to say theculture medium present in the fourth medium transfer zone 6 of the firstculture module m₁ overflows either via a tube or over the top of thewall of the fourth medium transfer zone 6 of the first culture module m₁in a pipe 11 which ends in a substantially central zone of a siphoncreated by said centrifugal pump which constitutes the mediumcirculation means according to the invention of the base module m₀.

In a variant of this embodiment, illustrated highly schematically inFIG. 4 and in detail in FIG. 9, the stacked modules m constitute theculture vessel. In this variant of FIG. 4, there can exist for examplethree kinds of module, base modules m₀, modules m₁; m_(2, . . . , n)comprising the four zones and a head module m_(x) (not shown). The basemodule m₀ or basal module m₀ comprises medium circulation means andassembly means, it is designed to engage the first assembly means 24 aof a four zones module m_(1, 2, . . . , n) as explained above, and toconstitute the bottom of the vessel. The head module m₁ is designed toengage the second assembly means 24 b of a four zones modulem_(1; 2 . . . n). The four zones module m_(1; 2, . . . , n) engaged bythe base module m₀ can be the same as that engaged by the head modulem_(t) or the four zones module m_(1; 2, . . . n) engaged by the basemodule m₀ can be the first in a series of four zones modulesm_(1; 2, . . . , n) and the one engaged by the head module m_(t) isconsequently the second four zones module in said series of four zonesmodules m_(1; 2, . . . , n).

This variant functions in the same way as that explained in detail forFIG. 3.

FIGS. 5 and 6 illustrate half of the basal part of the base module m₀.FIG. 5 is a cross-section view and FIG. 6 is a top view. As can be seen,the medium is designed to enter the base module through at least oneinlet in a substantially central area represented by the letter x in thefigures. The rotation axis of the magnetic device 7 passes through thiscentre x, whether it be real or virtual. When the circulation means arein operation, the magnetic device 7 is in rotation about its rotationaxis, the rotation thereof creates a siphon which sucks the mediumwithin the medium circulation means. The zone in which the magneticdevice is in rotation is confined by baffles or walls 25. In thisembodiment two baffles have been shown, but their number can be muchgreater, for example 3, 4, 5, 6, 8, 10, etc. The baffles will preferablybe disposed symmetrically on the circumference defined by the whole ofthis.

The spaces 13 between the baffles 25 are medium outlet orifices. Indeed,the medium is sucked by the siphon created by the rotation of themagnetic device and the medium is expelled towards the outside of thezone delimited by the baffles 25, through the orifices 13 between thebaffles. Since two baffles 25 have been shown, there are two mediumoutlet orifices in this embodiment but their number can be much greater,for example 3, 4, 5, 6, 8, 10, etc. As the baffles are preferablydisposed symmetrically, the locations of the medium outlets 13 are alsoadvantageously disposed symmetrically. When the medium is expelled bythe outlets 13, it ends up in the essentially circular zone 27.

In this embodiment, the basal part of the base module m₀ has in itorifices 20, substantially tubular in shape, which are orifices allowingfor example an introduction of gas or gas mixture, fresh medium,discharge of gas or mixture of gas, drawing off of medium, etc.

In addition, a recess 31 is provided for accessing these orifices 20from the outside, which makes it possible to connect these orifices to asupply of gas or gas mixture, fresh medium, etc.

FIG. 7 is a view in section of the top part of the base module m₀according to the invention and FIG. 8 is a view from above of this samepart. The top part comprises substantially tubular-shaped medium inletorifices 11. These inlet orifices 11 guide the medium coming from thefourth medium transfer zone 6 of the device in the siphon created by therotation of the magnetic device. When the magnetic device is inrotation, the medium situated in the essentially circumferential zone 27depicted in FIGS. 5 and 6 enters the perforation 26, said perforation 26being in communication with the conduits 28 enabling the expelled mediumto reach a zone 30 in medium communication with the first mediumtransfer zone 3 of the device, in particular with the essentiallytubular part of the first medium transfer zone.

The top part depicted in FIG. 7 is an element designed to be disposed onthe bottom part depicted in FIG. 5. Naturally, this base module rn₀could also be obtained in another way, but for reasons of simplicity ofproduction it has been produced for this embodiment in two parts thatcan be connected together. As can be seen moreover, the top part and thebottom part are connected together in a preferentially sealed manner atthe recesses 29 illustrated in the two FIGS. 5 and 7.

All the illustrations of medium circulation means of the presentapplication can also be produced in various ways. It goes without sayingthat all the ways for producing the various embodiments of the mediumcirculation means, confined or not in the base module, are included inthe scope of the claimed protection.

FIG. 9 is a cross section view of a particularly advantageous embodimentof a device according to the invention, whilst FIG. 10 is an explodedcross section view of the same embodiment. The exploded view gives aclear understanding of the particularly practical and inventive aspectof the present invention.

Consequently the two figures will be commented on at the same time. Ascan be seen, this embodiment of a device according to the inventionconsists, from bottom to top, of a clamped stack of

-   -   the bottom part of the base module m₀ (m_(0a)) comprising a zone        in which the magnetic device is in rotation which is confined by        baffles 25. The space 13 between the baffles 25 visible in this        figure is a medium outlet orifice. This is because the medium is        sucked by the siphon created by the rotation of the magnetic        device and the medium is expelled towards the outside 27        (essentially circular zone) of the zone delimited by the baffles        25 through the orifices 13 between the baffles 25,    -   the top part of the base module m₀(m₀b) comprising substantially        tubular shaped medium inlet orifices 11. These inlet orifices 11        guide the medium coming from the fourth medium transfer zone in        the siphon created by the rotation of the magnetic device 7.        When the magnetic device is in rotation, the medium situated in        the essentially circular zone 27 enters the perforation 26, said        perforation 26 being in communication with the conduits 28        enabling the expelled medium to reach a zone 30 in medium        communication with the first medium transfer zone 3, in        particular with the essentially tubular part of the first medium        transfer zone,    -   a first culture module m₁ as explained in detail in the        explanation of FIG. 3,    -   to a second culture module m₂ (see FIG. 3),    -   a head module comprising a recess 33 provided with an optical        sensor 23 immersed in the culture medium, a cover 19 comprising        fixing means 22 connected to a part 16 a of the top wall 16 of        the second culture zone 5 of the second culture module m₂.

All the modules comprise fixing means 24 a and 24 b as illustratedschematically in FIGS. 4, 9 and 10. Each module comprises several ofthese, which, according to the required assembly, will be used or not,but this makes it possible to obtain a single culture module which canbe assembled both with another culture module and with the base moduleor the head module. These fixing means are for example two concentriccircles provided with a circular seal, rapid connectors well known inthe art of cell culture, a screw pitch and a serration or any otherdevice for assembling these modules according to the invention.

In this embodiment, the basal part of the base module m₀ is bond withorifices 20 substantially tubular in shape which are orifices allowingin this case an introduction of gas or gas mixture. The gas inletorifice 20 is connected to a tube 32 which ends above the level of theculture medium, enabling the gas or gas mixture to reach at least afourth medium transfer zone 6 of the culture device 1 according to theinvention. All the ambient atmospheres of the fourth medium transferzone 6 are connected by similar tubes 32 so that the gas mixture canreach the top. It is particularly advantageous in a device with modulesstackable for height which can rise very high to provide a gaseoussupply through the bottom of the reactor.

In a variant, the basal part comprises a gas or gas mixture feed tubefor bringing the gaseous substance into the zone in which the magneticdevice is situated. In this way, the incoming gas is stirred by therotation of the magnetic device and the dissolution of the oxygen isimproved by the movement of the medium. The excess gas is also stirredand moves upwards again in the form of small bubbles. This variant isalso applicable to the embodiment illustrated in FIG. 1.

In addition, a recess 31 is provided for accessing these orifices 20from the outside, which makes it possible to connect these orifices to asupply of gas, gas mixture, fresh medium, etc.

The top part mob of the module_(m0) is an element designed to be grippedby virtue of the fixing means 28 and sealingly by virtue of the circularseal 34 on the bottom part m_(Oa) of the base module m₀.

Naturally the present invention is in no way limited to the embodimentsdescribed above and many modifications can be made thereto withoutdeparting from the scope of the accompanying claims.

For example, the embodiment of a device according to the inventiondepicted in FIG. 1 can also comprise a nutriment feed, either in a tubethrough the cover, or a tube through one of the walls of the device.Likewise, heating means can also be present in the first or fourth zoneof the device or of a module or each four zones module. Possibly, thedevice according to the invention can also comprise several mediumcirculation means, for example several centrifugal pumps.

The devices and methods of the present invention enable a homogenousflow of culture medium upon entry trough the orifices (17) in the bottompart (15) of the second zone (5) and consequently also during thefurther passage through this second zone (5). This in contrast todevices wherein the first zone (3) is in direct contact with the secondzone (5) which results in a non-homogenous flow throughout the secondzone (5). Such non-homogeneous flow results in the present ofundersupplied or dead zones within the cell culture zone which areinsufficiently supplied with oxygen and nutrients, and wherein cellgrowth and/or metabolism is far from optimal.

As described above in detail, a particular embodiment of the devices andmethods described in the present invention relates to devices and theiruse, wherein the third zone (4) is a zone internal to said second zone(5) and external to said first zone (3) as depicted in FIG. 1. The flowof the medium from the basal part of the first zone (3 a) upwards viathe top cylindrical part of the first zone (3 b), further downwards viathe cylindrical top part (overflow 37) of the third transfer zone (4 b)to the basal part of the third zone (4 a) generates the desiredhomogeneous flow upon entry in the bottom part of the second zone (5).

The presence of the cylindrical parts 3 b and 4 b further allows an easyaccess to the medium for assaying its properties prior to entry in thesecond zone (5). The presence of a cylindrical element 4 b also preventsthat a high pressure is built up in the device. The presence ofcylindrical parts 3 b and 4 b allows in addition the manufacture of adevice comprising different modules as depicted in FIG. 3.

Other embodiments of the devices and methods described in the presentinvention relates to devices as depicted in FIG. 1 which are modifiedsuch in that the liquid flow through the cylindrical parts 3 b and 4 bis bypassed. In these alternative embodiments of devices of the presentinvention the third zone (4) is a located entirely below the second zone(5) and entirely above the first zone (3). FIG. 11 shows an embodimentof a device wherein the part of the device corresponding to thecylindrical parts of the first zone and the second zone (3 b and 4 b) inFIG. 1 are replaced by a solid element (35) in e.g a plastic, glass ormetal.

In this device as depicted in FIG. 11 the first zone (3) and the secondzone (4) consist of a flattened shaped volume corresponding respectivelyto the basal parts 3 a and 4 a as depicted on Figure A and lack thecylindrical parts 3 b and 4 b, shown in FIG. 1.

With this adaptation the culture medium can equally overflow from thefirst zone (3) to the third zone (4), via the overflow 37. The flow ofthe medium created by the stirring device (7) is rendered homogeneous bythe separating wall between first zone (3) and third zone (4) andresults in a homogeneous flow upon entry of the second zone (5).

In particular embodiments, as indicated in FIG. 11, the separating wallbetween the first zone (3) and the third zone (4) consist of ahorizontal part as well as a of a vertical part, wherein this verticalpart with the overflow has a height of about up to 5%, up to 10%, up to20% or even up to 50% of the height of the third zone (4). In otherparticular embodiments, the vertical part of the separating wall betweenthe first zone (3) and the third zone (4) is absent.

In particular embodiments the solid element 35 is provided with channelsadapted to incorporate for example a probe to measure a condition of themedium in the third zone (4) (pH, oxygen, temperature, .). In otherparticular embodiments, solid element 35 is provided with a channelcomprising a safety pressure valve which can open when an excessivepressure is built up in the first zone (3) and third zone (4).

FIGS. 12 and 13 show yet alternative embodiments of a device wherein thecylindrical parts 3 b and 4 b of the respectively the first zone and thethird zone second zone are absent compare to FIG. 1. The volumepreviously occupied by elements 3 b and 4 b, now becomes parts of thesecond zone (5) resulting a in more efficient use of the deviceresulting from the enlarged volume which is suitable for cell growth.

In order to prevent the direct inflow of medium from the first zone (3)into the second zone (5) without an homogenous flow distribution intothe third zone (4), the orifices (17) in the bottom wall of the secondzone (5) are closed at those regions (36 b) which are located above anopening (36 a) in the wall between the first and the third zone. Theadaptation of the device by providing closed region 36 b above opening36 a results in the overflow of the culture medium from the firstculture medium transfer zone (3) into the third culture medium transferzone (4) before it enters as a homogenous flow into the second zone (5).

FIG. 12 shows a schematic representation of a device wherein one centralopening (36 a) and corresponding closed region (36 b) present. Inparticular embodiments, a plurality of openings (36 a) and correspondingclosed regions (36 b) is provided into respectively the separating wallbetween first zone (3) and third zone (4), and into the wall betweenthird zone (4) and second zone (5). Typically such plurality of openings(36 a) and corresponding closed regions (36 b) are distributedsymmetrically.

In an alternative embodiment of the present invention, the homogenousflow of the medium is achieved by providing an flow redistributingelement (36 c) within the third zone (4) as indicate in FIG. 13. Suchelement can have any shape suitable for an appropriate redistribution ofthe medium coming from the first zone (3) to obtain a homogenous liquidflow in third zone (3) prior to entry in the second zone (5).

In a particular embodiment element 36 c has the form of a set ofradially extending rods with a circular, diamond or oval cross section,positioned above corresponding radially applied openings (36 a).

LIST OF COMPONENTS

-   1. culture device-   2. culture vessel-   3. first culture medium transfer zone-   3 a basal part of the first culture medium transfer zone-   3 b top cylindrical part of the first culture medium transfer zone-   4. third culture medium transfer zone-   4 a basal part of the third culture medium transfer zone-   4 b cylindrical top part of the third culture medium transfer zone-   5. second culture zone-   6. fourth culture medium transfer zone-   7. magnetic device-   8. central rotation axis-   9. top engagement means-   10. bottom engagement means-   11. medium inlet-   12. diversion baffle-   13. medium outlet-   14. medium guiding means-   15. bottom wall of the second culture zone-   16. top wall of the second culture zone-   17. orifices in top and bottom walls of the second culture zone-   18. inclined wall-   19. cover of the culture vessel-   20. gas inlet orifice-   21. gas outlet orifice-   22. means of fixing the cover to the top wall of the second culture    zone-   23. sensors-   24. assembly means-   24 a. first assembly means-   24 b. second assembly means-   25. baffles or walls of the base module-   26. breakthrough in the base module-   27. essentially circular zone-   28. conduits-   29. recesses for the circular seal-   30. zone in medium communication with the first culture medium    transfer zone-   31. access recess to the orifices 20-   32. gas feeding tube-   33. recess in the cover for sensors-   34. circular seal-   35, solid element-   36 a. opening-   36 b. closed region-   36 c. flow redistributing element-   37. overflow-   m₀=base module-   m₁ . . . to m_(n)=culture modules-   m_(t)=head module-   M=culture medium-   C=cells-   D=top of the wall of the fourth culture zone

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

Other embodiments are within the scope of the claims.

1. A cell culture device comprising: a culture vessel provided with acover, wherein there is situated at least one first zone and at leastone second zone, wherein said first zone is a transfer zone for theculture medium essentially containing no cells and said second zone is acell culture zone, means of circulating the culture medium, allowingcirculation of the culture medium through the culture zone, said culturezone comprising a bottom wall and a top wall, wherein each wall isprovided with orifices allowing a transfer of culture medium essentiallyfree from cells, characterised in that: it also comprises at least onethird and at least one fourth zone, wherein both are culture mediumtransfer zones essentially free from cells, wherein said third zone isin medium communication with the first and second zone and wherein saidfourth zone is in medium communication with the second zone and with thefirst zone via the culture medium circulation means, and in that theculture medium circulation means allow a circulation of the culturemedium from bottom to top in said second culture zone.
 2. The deviceaccording to claim 1, wherein said third zone, has an overflow so thatthe culture medium overflows from the first culture medium transfer zoneto the third culture medium transfer zone.
 3. The device according toclaim 2, wherein said third zone is a zone internal to said second zoneand external to said first zone and said fourth zone is a zone externalto said second zone.
 4. The cell culture device according to claim 2,wherein the third zone is a zone located entirely below the second zoneand entirely above the first zone.
 5. The cell culture device accordingto claim 3, further comprising flow redistributing elements in the thirdzone or closed regions in the bottom wall of the second zone forproviding a homogenous flow of medium into said second zone.
 6. The cellculture device according to claim 3, comprising a series of culturemodules (m₁, . . . , m_(n)), each module comprising said first zone,said second zone, said third zone and said fourth zone, and wherein theadjacent modules in said series of culture modules are in mediumcommunication, and said first zone and said fourth zone of each moduleare in communication with said circulation means, directly orindirectly.
 7. The cell culture device according to claim 6, wherein thecirculation means are confined in a base module (m₀), and wherein saidbase module (m₀) is in medium communication with at least one firstmedium transfer zone and at least one fourth medium transfer zone,directly or indirectly.
 8. The cell culture device according to claim 6,further comprising a head module (m_(t)), said head module (m_(t))comprising at least the cover.
 9. The cell culture device according toclaim 1, wherein said at least one fourth zone comprises at least oneessentially vertical or inclined flow wall.
 10. The cell culture deviceaccording to claim 1, wherein said essentially vertical or inclined flowwall comprises a hydrophilic membrane.
 11. The cell culture deviceaccording to claim 1, further comprising at least one gas inlet orificeand one gas outlet orifice.
 12. The cell culture device according toclaim 1, wherein the cover of the culture vessel is connected to atleast part of said top wall of at least one second culture zone.
 13. Thecell culture device according to claim 6, wherein the circulation meansare confined in a base module (m₀), wherein said base module (m₀) is inmedium communication with at least one first medium transfer zone and atleast one fourth medium transfer zone, directly or indirectly, whereineach culture module (m₀, . . . , m_(n)) comprises in its top part afirst fixing means and in its bottom part a second fixing means andwherein said base module (m₀) also comprises in a top part (m_(0b)) afirst fixing means and said head module (m_(t)) also comprises in itsbottom part a second fixing means, wherein said first fixing means andsaid second fixing means are complementary fixing means for producing astacking sequence from bottom to top of a base module (m₀), at least oneculture module (m₀, . . . , m_(n)) and a head module (m_(t)).
 14. Thedevice according to claim 13, wherein said first and said second fixingmeans comprise means for producing said stacking sequence in a gas- andliquid-tight manner.
 15. A method of culturing cells in a culture vesselwith culture medium circulation, comprising the steps of: introductionof culture medium (M) into culture medium circulation means, dischargeof culture medium (M) from said culture medium circulation means, atleast one first transfer of culture medium (M) into a first culturemedium upward transfer zone, at least one second transfer of culturemedium (M) into a second cell culture zone, characterised in that italso comprises: at least one third culture medium transfer subsequent tosaid first culture medium transfer into a third culture medium transferzone, by overflowing from the first culture medium transfer zone to thethird culture medium transfer zone, at least one fourth culture mediumtransfer subsequent to said second culture medium transfer into a fourthculture medium transfer zone, by overflowing from the second culturezone into the fourth culture medium transfer zone, and in that saidsecond culture medium transfer is a downward culture medium transfer.16. The method of culturing cells according to claim 15, also comprisingoxygenation of the culture medium during one or more of said transfers.17. The method of culturing cells according to claim 15, wherein theoxygenation is carried out during the fourth culture medium transfer,and wherein said fourth culture medium transfer is a flow of saidculture medium (M) along a flow wall.
 18. The method of culturing cellsaccording to claim 15, wherein said flow of the culture medium (M) is aflow along a hydrophilic wall.